For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 4

Experiment 4
Isolation and Purification of Proteins

The isolation of proteins from the chemical mass or biological fluid in which they are found
normally requires the liberation of the cell contents by disruption of the tissue in a suitable
medium. The gentlest possible method for breaking open the cell is generally desirable. Water,
dilute salt, acid, or alkaline solutions may be used to extract the proteins. Association of proteins
C H E M 133 L B I O C H E M I S T R Y (Laboratory)

with other organic substances may prevent the extraction of the desired protein with aqueous
solvents. This requires preliminary treatment of a ground tissue suspension with weak detergent
solutions or organic solvents such as acetone, ether, or butanol.

PROCEDURE
Isolation of Casein from Milk

Prepare 25 mL of whole milk by diluting evaporated milk 1:1 or skim milk 1:3 by volume
using tap water. Heat the milk to 40°C and slowly over a 10-minute period, add 10% HCl drop
by drop, with thorough stirring, until a flocculent precipitate forms. The pH must decrease to
about 4.8. Avoid excess acid. Centrifuge the mixture and decant or pipette out the whey. Wash
the curd by re-suspending in 15 mL distilled water, stirring vigorously and decanting. Filter and
transfer the moist residue to a beaker. Add 10 mL distilled water and stir vigorously. Filter and
repeat the washing until the filtrate is chloride free. (Test a portion of the filtrate with 1%
AgNO3.)

NOTE: Some of the casein may be lost due to peptization while washing repeatedly. Adding a
drop or two of dilute HNO3 to wash water may help avoid this.

Dry the curd between sheets of filter paper and wash by suspending in enough 95%
alcohol to cover and stirring vigorously for 3 minutes. Filter and press the precipitate between
filter papers to remove the alcohol. Likewise wash with enough acetone and filter. Press the
precipitate between filter papers to remove the acetone and air dry. Weigh the dry crude
extract and calculate yield in g/mL.
 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 4

Group Members: Group No.

1. Section:
2.
Date Submitted:

Expt. 2 Isolation and Purification of Proteins

C H E M 133 L B I O C H E M I S T R Y (Laboratory)

I. Objectives Member 1 Member 2

Pre-lab Data
Data and Observations
Lab Report
Sub-total
Online Act Participation
Total Score

II. Apparatus: III. Symbols of Chemicals:

IV. Procedure: (Note: Write schematic diagram on the sheet provided.)

V. Data and Observations

Casein
Volume of milk with brand

Weight of protein isolated

Description of precipitate

Casein yield (g/mL)

 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 4

VI. Analysis: Guide Questions

Use the following questions as your guide in making your analysis. Note that your analysis are not just
answers to these questions simply placed in paragraph form. Please refer to the guide in making a lab
report as you make your lab analysis.

1. What principle is involved in the isolation of casein?

2. Why should HCl be added slowly to the milk with thorough stirring? What will be the effect if too
much acid has been added?
3. Explain the isoelectric isolation and purification of protein.

VII. Conclusion

Note: 1. Follow the designated format in making Lab Report

2. Place your Lab Analysis after Schematic Diagram
 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 4

Schematic Diagram of Procedure

THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO 4

 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 4

THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO 5

 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 4

Laboratory Report on Experiment 4

Isolation and Purification of Proteins

THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO 6